Histone methyltransferases have repeatedly been identified by genomics efforts as amplified, translocated, overexpressed, and mutated in a variety of cancers and other diseases, making this class of enzymes attractive candidates for therapeutic intervention. Despite these observations, the development of selective and potent inhibitors of this enzyme class remains in its infancy. The NSD histone methyltransferases, which are selective for histone H3 lysine 36 (H3K36) are frequently altered in various cancers. In particular, translocations of NSD2 leading to its overexpression are present in 15% of multiple myelomas, and available data suggest the catalytic ability of NSD2 promotes proliferation in myeloma cell lines and cancer cell lines derived from several other lineages. No selective inhibitors of NSD2 have been reported. We aim to collaborate with the MLPCN to identify and optimize at least one small-molecule inhibitor of NSD2. A high-throughput primary assay is in place and has been employed in the screening of a library of 1,200 small-molecules. A suite of secondary assays capable of identifying hits that inhibit NSD2 in cells is in place or undergoing final validation. Moreover, multiple high-throughput tertiary assays are in place to establish the in vitro selectivity of hit compounds for NSD2 over a range of methyltransferases. A lower- throughput cell-based tertiary assay to measure the impact of compound treatment on global histone lysine methylation at all sites simultaneously is also in place. It is anticipated that optimized probes will become highly valuable reagents in the epigenetics and cancer biology communities. We plan to measure the impact of optimized probes on proliferation in relevant cellular contexts to assess whether NSD2 inhibition may represent a promising therapeutic intervention. In particular, a panel of multiple myeloma cell lines harboring translocations of NSD2 will be employed, and a variety of cell lines that overexpress NSD2 from diverse lineages will also be tested. The impact of optimized probes on transcriptional regulation will be explored as well: as Wnt and androgen receptor signaling have previously been shown to be promoted by NSD family members, these pathways represent promising options.